Antibacterial 4,6-substituted 6&#39;, 6&#34; and 1 modified aminoglycoside analogs

ABSTRACT

The present invention is directed to analogs of aminoglycoside compounds as well as their preparation and use as prophylactic or therapeutics against microbial infection.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International PCT PatentApplication No. PCT/US2009/056391, which was filed on Sep. 9, 2009, nowpending, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/095,670, filed Sep. 10, 2008,which applications are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention is directed to novel aminoglycoside compounds andsynthetic methods for their preparation and use as therapeutic orprophylactic agents.

BACKGROUND OF THE INVENTION

A particular interest in modern drug discovery is the development ofnovel low molecular weight orally-bioavailable drugs that work bybinding to RNA. RNA, which serves as a messenger between DNA andproteins, was thought to be an entirely flexible molecule withoutsignificant structural complexity. Recent studies have revealed asurprising intricacy in RNA structure. RNA has a structural complexityrivaling proteins, rather than simple motifs like DNA. Genome sequencingreveals both the sequences of the proteins and the mRNAs that encodethem. Since proteins are synthesized using an RNA template, suchproteins can be inhibited by preventing their production in the firstplace by interfering with the translation of the mRNA. Since bothproteins and the RNAs are potential drug targeting sites, the number oftargets revealed from genome sequencing efforts is effectively doubled.These observations unlock a new world of opportunities for thepharmaceutical industry to target RNA with small molecules.

Classical drug discovery has focused on proteins as targets forintervention. Proteins can be extremely difficult to isolate and purifyin the appropriate form for use in assays for drug screening. Manyproteins require post-translational modifications that occur only inspecific cell types under specific conditions. Proteins fold intoglobular domains with hydrophobic cores and hydrophilic and chargedgroups on the surface. Multiple subunits frequently form complexes,which may be required for a valid drug screen. Membrane proteins usuallyneed to be embedded in a membrane to retain their proper shape. Thesmallest practical unit of a protein that can be used in drug screeningis a globular domain. The notion of removing a single alpha helix orturn of a beta sheet and using it in a drug screen is not practical,since only the intact protein may have the appropriate 3-dimensionalshape for drug binding. Preparation of Biologically Active Proteins forScreening is a Major Limitation in Classical high throughput screening.Quite often the limiting reagent in high throughput screening efforts isa biologically active form of a protein which can also be quiteexpensive.

For screening to discover compounds that bind RNA targets, the classicapproaches used for proteins can be superceded with new approaches. AllRNAs are essentially equivalent in their solubility, ease of synthesisor use in assays. The physical properties of RNAs are independent of theprotein they encode. They may be readily prepared in large quantitythrough either chemical or enzymatic synthesis and are not extensivelymodified in vivo. With RNA, the smallest practical unit for drug bindingis the functional subdomain. A functional subdomain in RNA is a fragmentthat, when removed from the larger RNA and studied in isolation, retainsits biologically relevant shape and protein or RNA-binding properties.The size and composition of RNA functional subdomains make themaccessible by enzymatic or chemical synthesis. The structural biologycommunity has developed significant experience in identification offunctional RNA subdomains in order to facilitate structural studies bytechniques such as NMR spectroscopy. For example, small analogs of thedecoding region of 16S rRNA (the A-site) have been identified ascontaining only the essential region, and have been shown to bindantibiotics in the same fashion as the intact ribosome.

The binding sites on RNA are hydrophilic and relatively open as comparedto proteins. The potential for small molecule recognition based on shapeis enhanced by the deformability of RNA. The binding of molecules tospecific RNA targets can be determined by global conformation and thedistribution of charged, aromatic, and hydrogen bonding groups off of arelatively rigid scaffold. Properly placed positive charges are believedto be important, since long-range electrostatic interactions can be usedto steer molecules into a binding pocket with the proper orientation. Instructures where nucleobases are exposed, stacking interactions witharomatic functional groups may contribute to the binding interaction.The major groove of RNA provides many sites for specific hydrogenbonding with a ligand. These include the aromatic N7 nitrogen atoms ofadenosine and guanosine, the O4 and O6 oxygen atoms of uridine andguanosine, and the amines of adenosine and cytidine. The rich structuraland sequence diversity of RNA suggests to us that ligands can be createdwith high affinity and specificity for their target.

Although our understanding of RNA structure and folding, as well as themodes in which RNA is recognized by other ligands, is far from beingcomprehensive, significant progress has been made in the last decade(Chow, C. S.; Bogdan, F. M., Chem. Rev., 1997, 97, 1489, Wallis, M. G.;Schroeder, R., Prog. Biophys. Molec. Biol. 1997, 67, 141). Despite thecentral role RNA plays in the replication of bacteria, drugs that targetthese pivotal RNA sites of these pathogens are scarce. The increasingproblem of bacterial resistance to antibiotics makes the search fornovel RNA binders of crucial importance.

Certain small molecules can bind and block essential functions of RNA.Examples of such molecules include the aminoglycoside antibiotics anddrugs such as erythromycin which binds to bacterial rRNA and releasespeptidyl-tRNA and mRNA. Aminoglycoside antibiotics have long been knownto bind RNA. They exert their antibacterial effects by binding tospecific target sites in the bacterial ribosome. For the structurallyrelated antibiotics neamine, ribostamycin, neomycin B, and paromomycin,the binding site has been localized to the A-site of the prokaryotic 16Sribosomal decoding region RNA (Moazed, D.; Noller, H. F., Nature, 1987,327, 389). Binding of aminoglycosides to this RNA target interferes withthe fidelity of mRNA translation and results in miscoding andtruncation, leading ultimately to bacterial cell death (Alper, P. B.;Hendrix, M.; Sears, P.; Wong, C., J. Am. Chem. Soc., 1998, 120, 1965).

There is a need in the art for new chemical entities that work againstbacteria with broad-spectrum activity. Perhaps the biggest challenge indiscovering RNA-binding antibacterial drugs is identifying vitalstructures common to bacteria that can be disabled by small moleculedrug binding. A challenge in targeting RNA with small molecules is todevelop a chemical strategy which recognizes specific shapes of RNA.There are three sets of data that provide hints on how to do this:natural protein interactions with RNA, natural product antibiotics thatbind RNA, and man-made RNAs (aptamers) that bind proteins and othermolecules. Each data set, however, provides different insights to theproblem.

Several classes of drugs obtained from natural sources have been shownto work by binding to RNA or RNA/protein complexes. These include threedifferent structural classes of antibiotics: thiostreptone, theaminoglycoside family and the macrolide family of antibiotics. Theseexamples provide powerful clues to how small molecules and targets mightbe selected. Nature has selected RNA targets in the ribosome, one of themost ancient and conserved targets in bacteria. Since antibacterialdrugs are desired to be potent and have broad-spectrum activity theseancient processes fundamental to all bacterial life represent attractivetargets. The closer we get to ancient conserved functions the morelikely we are to find broadly conserved RNA shapes. It is important toalso consider the shape of the equivalent structure in humans, sincebacteria were unlikely to have considered the therapeutic index of theirRNAs while evolving them.

A large number of natural antibiotics exist, these include theaminoglycosides, kirromycin, neomycin, paromomycin, thiostrepton, andmany others. They are very potent, bactericidal compounds that bind RNAof the small ribosomal subunit. The bactericidal action is mediated bybinding to the bacterial RNA in a fashion that leads to misreading ofthe genetic code. Misreading of the code during translation of integralmembrane proteins is thought to produce abnormal proteins thatcompromise the barrier properties of the bacterial membrane.

Antibiotics are chemical substances produced by various species ofmicroorganisms (bacteria, fungi, actinomycetes) that suppress the growthof other microorganisms and may eventually destroy them. However, commonusage often extends the term antibiotics to include syntheticantibacterial agents, such as the sulfonamides, and quinolines, that arenot products of microbes. The number of antibiotics that have beenidentified now extends into the hundreds, and many of these have beendeveloped to the stage where they are of value in the therapy ofinfectious diseases. Antibiotics differ markedly in physical, chemical,and pharmacological properties, antibacterial spectra, and mechanisms ofaction. In recent years, knowledge of molecular mechanisms of bacterial,fungal, and viral replication has greatly facilitated rationaldevelopment of compounds that can interfere with the life cycles ofthese microorganisms.

At least 30% of all hospitalized patients now receive one or morecourses of therapy with antibiotics, and millions of potentially fatalinfections have been cured. At the same time, these pharmaceuticalagents have become among the most misused of those available to thepracticing physician. One result of widespread use of antimicrobialagents has been the emergence of antibiotic-resistant pathogens, whichin turn has created an ever-increasing need for new drugs. Many of theseagents have also contributed significantly to the rising costs ofmedical care.

When the antimicrobial activity of a new agent is first tested a patternof sensitivity and resistance is usually defined. Unfortunately, thisspectrum of activity can subsequently change to a remarkable degree,because microorganisms have evolved the array of ingenious alterationsdiscussed above that allow them to survive in the presence ofantibiotics. The mechanism of drug resistance varies from microorganismto microorganism and from drug to drug.

The development of resistance to antibiotics usually involves a stablegenetic change, heritable from generation to generation. Any of themechanisms that result in alteration of bacterial genetic compositioncan operate. While mutation is frequently the cause, resistance toantimicrobial agents may be acquired through transfer of geneticmaterial from one bacterium to another by transduction, transformationor conjugation.

One group has prepared and assayed 6′-N-methyl and 6′-N-ethylderivatives of amikacin (Umezawa, et al., Journal of Antiboiotics, 1975,28(6), 483-485). They showed that these derivatives were hardly affectedby the 6′-N-acetyltransferase which has been shown to inactivateamikacin.

For the foregoing reasons, there is a need for new chemical entitiesthat possess antimicrobial activity. Further, in order to accelerate thedrug discovery process, new methods for synthesizing aminoglycosideantibiotics are needed to provide an array of compounds that arepotentially new drugs for the treatment microbial infections.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds having formulaI:

wherein:

-   -   each R₁ and R₂ is, independently, H or an amino protecting        group;    -   each R₃ is, independently, H or a hydroxyl protecting group;    -   each R₄ and R₅ is independently, H, amino protecting group,        C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,        substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl or substituted C₂-C₁₂        alkynyl;    -   Q₁ is hydroxyl, a protected hydroxyl, cyano, azido, or NR₈R₉;    -   Q₂ is NH₂ or N(CH₂R₆)R₇;        -   R₆ and R₇ are each, independently, H, C₁-C₁₂ alkyl,            substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical,            substituted C₇-C₉ alicyclic radical, C₂-C₁₂ alkenyl,            substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl or substituted            C₂-C₁₂ alkynyl;        -   R₈ and R₉ are each, independently, H, CN, C₁-C₁₂ alkyl,            substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical,            substituted C₇-C₉ alicyclic radical, C₂-C₁₂ alkenyl,            substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl or substituted            C₂-C₁₂ alkynyl;            -   wherein said substituted groups are mono or poly                substituted with optionally protected substituent groups                selected from halogen, trifluoromethyl, cyano, OJ₃,                NJ₁J₂, C(═O)—NJ₁J₂, N(H)C(═O)-J₁, N(J₁)-(CH₂)_(n)—OJ₃,                N(J₁)-(CH₂)_(n)—NJ₁J₂, C₅-C₂₀ aryl, substituted C₅-C₂₀                aryl, C₇-C₉ alicyclic radical, substituted C₇-C₉                alicyclic radical, heterocycle radical, substituted                heterocycle radical, heteroaryl, substituted heteroaryl,                azido, carboxy, acyl (C(═O)—X), ═O, cyano, sulfonyl                (S(═O)₂—X) and sulfoxyl (S(═O)—X);                -   each X is, independently, H, C₁-C₁₂ alkyl or                    substituted C₁-C₁₂ alkyl;                -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl,                    substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,                    substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,                    substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted                    C₅-C₂₀ aryl, acyl (C(═O)—X), a heterocycle radical,                    a substituted heterocycle radical, heteroaryl or                    substituted heteroaryl;                -   each J₃ is, independently, H, C₁-C₁₂ alkyl,                    substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,                    substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,                    substituted C₂-C₁₂ alkynyl, C₁-C_(u) aminoalkyl,                    substituted C₁-C₁₂ aminoalkyl or a hydroxyl                    protecting group;                -   n is from 1 to 20; and    -   with the proviso that when Q₁ is hydroxyl or protected hydroxyl,        Q₂ is N(CH₂R₆)R₇ and R₇ is H then R₆ is other than H or methyl.

In one embodiment the substituent groups are independently selected fromOH, NH₂, N(H)alkyl, amide (C(═O)—N(H)J₂ or N(H)C(═O)-J₁),N(J₁)-(CH₂)_(n)—OJ₃, N(J₁)-(CH₂)_(n)—NJ₁J₂, C₇-C₉ alicyclic radical,C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, a heterocycle radical, asubstituted heterocycle radical, heteroaryl or substituted heteroaryl.

In one embodiment each R₁ and R₂ is H. In a further embodiment each R₃is H. In another embodiment Q₁ is hydroxyl. In another embodiment Q₁ isNR₈R₉.

In one embodiment one of R₈ and R₉ is H and the other of R₈ and R₉ isC₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical orsubstituted C₇-C₉ alicyclic radical. In a further embodiment one of R₈and R₉ is H and the other of R₈ and R₉ is substituted C₁-C₁₂ alkylwherein each of the substituents is, independently, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, C₇-C₉ alicyclic radical, substituted C₇-C₉alicyclic radical, heterocycle radical, substituted heterocycle radical,heteroaryl or substituted heteroaryl.

In one embodiment Q₁ is NH₂. In a further embodiment Q₁ is NH₂ and R₇ isH or C₁-C₁₂ alkyl. In another embodiment Q₁ is NH₂ and R₆ is C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical or substitutedC₇-C₉ alicyclic radical. In a further embodiment Q₁ is NH₂ and R₆ issubstituted C₁-C₁₂ alkyl wherein each of the substituents is,independently, C₇-C₉ alicyclic radical, substituted C₇-C₉ alicyclicradical, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical,substituted heterocycle radical, heteroaryl or substituted heteroaryl.In a further embodiment Q₁ is NH₂ and R₆ is substituted C₁-C₁₂ alkylwherein each of said substituents is, independently, C₅-C₂₀ aryl orsubstituted C₅-C₂₀ aryl.

In one embodiment R₁, R₄ and R₅ are each H.

In one embodiment one of R₄ and R₅ is H and the other of R₄ and R₅ isC₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl. In a further embodiment one ofR₄ and R₅ is H and the other of R₄ and R₅ is C₁-C₁₂ alkyl or substitutedC₁-C₁₂ alkyl and each substituent group is, independently, C₅-C₉alicyclic radical, substituted C₇-C₉ alicyclic radical, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl or substituted heteroaryl. In another embodiment oneof R₄ and R₅ is H and the other of R₄ and R₅ is C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl and the other of R₄ and R₅ is substitutedC₁-C₁₂ alkyl wherein each substituent group is, independently, C₅-C₂₀aryl or substituted C₅-C₂₀ aryl.

In one embodiment each of R₁, R₄ and R₅ are H. In a further embodimenteach of R₁, R₄ and R₅ are H and Q₁ is OH. In another embodiment each ofR₁, R₄ and R₅ are H, Q₁ is OH and R₇ is H. In a further embodiment eachof R₁, R₄ and R₅ are H, Q₁ is OH, R₇ is H and R₆ is CH₂—C₆H₅, CH(CH₃)₂or (CH₂)₂—C₆H₅. In another embodiment each of R₁, R₄ and R₅ are H, Q₁ isOH and R₆ is H and R₇ is CH₃. In a further embodiment each of R₁, R₄ andR₅ are H and Q₂ is NH₂. In another embodiment each of R₁, R₄ and R₅ areH, Q₂ is NH₂ and R₈ is H. In a further embodiment each of R₁, R₄ and R₅are H, Q₂ is NH₂, R₈ is H and R₉ is cyclohexyl (C₆H₁₁), (CH₂)₂—C₆H₅,CH(CH₃)₂ or CH₃.

In one embodiment Q₁ is azido or cyano. In a further embodiment Q₁ isazido or cyano and Q₂ is N(CH₂R₆)R₇ and R₇ is H or C₁-C₁₂ alkyl. Inanother embodiment Q₁ is azido or cyano and Q₂ is amino. In a furtherembodiment Q₁ is azido or cyano and Q₂ is amino and R₁, R₄ and R₅ areeach H.

The present invention also provides compounds that have specificstereochemistry about chiral centers having the configuration:

The present invention also provides methods of using a compound of theinvention in therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides aminoglycoside compounds having theformula I:

wherein:

-   -   each R₁ and R₂ is, independently, H or an amino protecting        group;    -   each R₃ is, independently, H or a hydroxyl protecting group;    -   each R₄ and R₅ is independently, H, amino protecting group,        C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,        substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl or substituted C₂-C₁₂        alkynyl;    -   Q₁ is hydroxyl, a protected hydroxyl, cyano, azido, or NR₈R₉;    -   Q₂ is NH₂ or N(CH₂R₆)R₇;        -   R₆ and R₇ are each, independently, H, C₁-C₁₂ alkyl,            substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical,            substituted C₇-C₉ alicyclic radical, C₂-C₁₂ alkenyl,            substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl or substituted            C₂-C₁₂ alkynyl;        -   R₈ and R₉ are each, independently, H, CN, C₁-C₁₂ alkyl,            substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical,            substituted C₇-C₉ alicyclic radical, C₂-C₁₂ alkenyl,            substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl or substituted            C₂-C₁₂ alkynyl;            -   wherein said substituted groups are mono or poly                substituted with optionally protected substituent groups                selected from halogen, trifluoromethyl, cyano, OJ₃,                NJ₁J₂, C(═O)—NJ₁J₂, N(H)C(═O)-J₁, N(J₁)-(CH₂)_(n)—OJ₃,                N(J₁)-(CH₂)_(n)—NJ₁J₂, C₅-C₂₀ aryl, substituted C₅-C₂₀                aryl, C₇-C₉ alicyclic radical, substituted C₇-C₉                alicyclic radical, heterocycle radical, substituted                heterocycle radical, heteroaryl, substituted heteroaryl,                azido, carboxy, acyl (C(═O)—X), ═O, cyano, sulfonyl                (S(═O)₂—X) and sulfoxyl (S(═O)—X);                -   each X is, independently, H, C₁-C₁₂ alkyl or                    substituted C₁-C₁₂ alkyl;                -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl,                    substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,                    substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,                    substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted                    C₅-C₂₀ aryl, acyl (C(═O)—X), a heterocycle radical,                    a substituted heterocycle radical, heteroaryl or                    substituted heteroaryl;                -   each J₃ is, independently, H, C₁-C₁₂ alkyl,                    substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,                    substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,                    substituted C₂-C₁₂ alkynyl, C₁-C₁₂ aminoalkyl,                    substituted C₁-C₁₂ aminoalkyl or a hydroxyl                    protecting group;                -   n is from 1 to 20; and    -   with the proviso that when Q₁ is hydroxyl or protected hydroxyl,        Q₂ is N(CH₂R₆)R₇ and R₇ is H then R₆ is other than H or methyl.

In a preferred embodiment the compounds of the present invention areprepared from Amikacin free base which is orthogonally protected andreacted with reactive groups to place functional groups on either 6′ or6″-position. Amikacin is available from a number of commercial sourcesas the HCl salt and is subsequently converted to the free base. Themethods are amenable to a wide variety of chemical reactions to preparea large number of Amikacin analogs. In some preferred embodiments of thepresent invention each R₁, R₂, R₃, R₄ and R₅ is H and one of Q₁ and Q₂is substituted with a variety of functional groups while Q₁ is hydroxylor Q₂ is amino.

The term “alkyl,” as used herein, refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred. The term “lower alkyl” asused herein includes from 1 to about 6 carbon atoms. Alkyl groups asused herein may optionally include one or more further substitutentgroups (see substituent group list below).

The term “alkenyl,” as used herein, refers to a straight or branchedhydrocarbon chain radical containing up to twenty four carbon atomshaving at least one carbon-carbon double bond. Examples of alkenylgroups include, but are not limited to, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.Alkenyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkenyl groups as used herein may optionallyinclude one or more further substitutent groups.

The term “alkynyl,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms and havingat least one carbon-carbon triple bond. Examples of alkynyl groupsinclude, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and thelike. Alkynyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkynyl groups as used herein mayoptionally include one or more further substitutent groups.

The term “aminoalkyl” as used herein, refers to an amino substitutedalkyl radical. This term is meant to include C₁-C₁₂ alkyl groups havingan amino substituent at any position and wherein the alkyl groupattaches the aminoalkyl group to the parent molecule. The alkyl or aminoportions of the aminoalkyl group can be further substituted withsubstituent groups.

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interrupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interrupted by heteroatoms include without limitationpolyalkoxys, such as polyalkylene glycols, polyamines, and polyimines,for example. Aliphatic groups as used herein may optionally includefurther substitutent groups.

The term “alicyclic” refers to a cyclic ring system wherein the ring isaliphatic. The ring system can comprise one or more rings and wherein atleast one ring is aliphatic. Alicyclics include rings having any degreeof saturation. Preferred alicyclics include rings having from about 5 toabout 9 carbon atoms in the ring. Alicyclic as used herein mayoptionally include further substitutent groups.

The term “alkoxy,” as used herein, refers to a radical formed between analkyl group and an oxygen atom wherein the oxygen atom is used to attachthe alkoxy group to a parent molecule. Examples of alkoxy groupsinclude, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy andthe like. Alkoxy groups as used herein may optionally include furthersubstitutent groups.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “aryl” and “aromatic,” as used herein, refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include, but are not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstitutent groups.

The terms “aralkyl” and “arylalkyl,” as used herein, refer to a radicalformed between an alkyl group and an aryl group wherein the alkyl groupis used to attach the aralkyl group to a parent molecule. Examplesinclude, but are not limited to, benzyl, phenethyl and the like. Aralkylgroups as used herein may optionally include further substitutent groupsattached to the alkyl, the aryl or both groups that form the radicalgroup.

The term “heterocyclic,” or “heterocyclic radical” as used herein,refers to a radical mono-, or poly-cyclic ring system that includes atleast one heteroatom and is unsaturated, partially saturated or fullysaturated, thereby including heteroaryl groups. Heterocyclic is alsomeant to include fused ring systems wherein one or more of the fusedrings contain no heteroatoms. A heterocyclic group typically includes atleast one atom selected from sulfur, nitrogen or oxygen. Examples ofheterocyclic groups include, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and thelike. Heterocyclic groups as used herein may optionally include furthersubstitutent groups.

The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes a heteroatom. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals can be attached to a parent molecule directlyor through a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstitutent groups.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined, attached to a parent molecule via an alkyl group.Examples include, but are not limited to, pyridinylmethyl,pyrimidinylethyl and the like. Heteroarylalkyl groups as used herein mayoptionally include further substitutent groups.

The term “acyl,” as used herein, refers to a radical formed by removalof a hydroxyl group from an organic acid and has the general formula—C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examplesinclude aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls,aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substitutent groups.

The terms “substituent and substituent group,” as used herein, are meantto include groups that are typically added to other groups or parentcompounds to enhance desired properties or give desired effects.Substituent groups can be protected or unprotected and can be added toone available site or to many available sites in a parent compound.Substituent groups may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to the parent compound. Suchgroups include without limitation, halogen, hydroxyl, alkyl, alkenyl,alkynyl, acyl (—C(O)R_(a)), carboxyl (—C(O)O—R_(a)), aliphatic,alicyclic, alkoxy, substituted oxo (—O—R_(a)), aryl, aralkyl,heterocyclic, heteroaryl, heteroarylalkyl, amino (—NR_(b)R_(c)),imino(═NR_(b)), amido (—C(O)NR_(b)R_(c) or —N(R_(b))C(O)R_(a)), azido(—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)NR_(b)R_(c) or—N(R_(b))C(O)OR_(a)), ureido (—N(R_(b))C(O)NR_(b)R_(c)), thioureido(—N(R_(b))C(S)NR_(b)R_(c)), guanidinyl (—N(R_(b))C(═NR_(b))NR_(b)R_(c)),amidinyl (—C(═NR_(b))NR_(b)R_(c) or —N(R_(b))C(NR_(b))R_(a)), thiol(—SR_(b)), sulfinyl (—S(O)R_(b)), sulfonyl (—S(O)₂R_(b)) andsulfonamidyl (—S(O)₂NR_(b)R_(c) or —N(R_(b))S(O)₂R_(b)). Wherein eachR_(a), R_(b) and R_(c) is a further substituent group with a preferredlist including without limitation alkyl, alkenyl, alkynyl, aliphatic,alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic andheteroarylalkyl.

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively and/or orthogonally to protect sites during reactionsat other reactive sites and can then be removed to leave the unprotectedgroup as is or available for further reactions. Protecting groups asknown in the art are described generally in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York(1999).

Examples of hydroxyl protecting groups include, but are not limited to,benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl (BOC),isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(tri-methylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl (Alloc), acetyl (Ac), formyl,chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl(Bz), methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilyl ethyl,1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl (Bn),para-methoxybenzyldiphenylmethyl, triphenylmethyl (trityl),4,4′-dimethoxytriphenylmethyl (DMT), substituted or unsubstituted9-(9-phenyl)xanthenyl (pixyl), tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxyl protecting groups for the present invention are DMTand substituted or unsubstituted pixyl.

Examples of amino protecting groups include, but are not limited to,t-butoxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (Fmoc),benzyloxycarbonyl, and the like.

Examples of thiol protecting groups include, but are not limited to,triphenylmethyl (Trt), benzyl (Bn), and the like.

Included within the scope of the present invention are thepharmaceutically acceptable salts of the foregoing compounds. As usedherein, the term “pharmaceutically acceptable salts” refers to non-toxicacid addition salts and alkaline earth metal salts of the compounds ofthe invention. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, orseparately by reacting the free base or acid functions with a suitableorganic acid or base. Representative acid addition salts include thehydrochloride, hydrobromide, sulphate, bisulphate, acetate, oxalate,valerate, oleate, palmitate, stearate, laurate, borate, benzoate,lactate, phosphate, tosylate, mesylate, citrate, maleate, fumarate,succinate, tartrate, glucoheptonate, lactobionate, lauryl sulfate saltsand the like. Representative alkali or alkaline earth metal saltsinclude the sodium, calcium, potassium and magnesium salts.

As per MIC assays performed using some preferred compounds of theinvention it has been found that the compounds possess antibacterialactivity against at least P aeruginosa 29248, P aeruginosa 27853, Paeruginosa 25416, P. vulgaris 8427, A. bauman11 WR-2, E. coli 25922 andS. aureus 13709. The compounds of the invention are therefore useful inantibiotic treatments. In addition, the compounds, by reason of their invitro activity, may be used in scrub solutions for surface inhibition ofbacterial growth e.g. in sterilization of glasswear or as an additive infabric laundering compositions.

Susceptible organisms generally include those gram positive and gramnegative, aerobic and anaerobic organisms whose growth can be inhibitedby the compounds of the invention such as Staphylococcus, Lactobacillus,Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella,Pseudomonas, Acinetobacter, Proteus, Campylobacter, Citrobacter,Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium, Salmonella,Shigella, Serratia, Haemophilus, Brucella and other organisms.

EXAMPLES

NMR: NMR spectra were recorded on a 300 M Hz Bruker.

LCMS Method A: A 4×125 mm 5 uM Polar RP column using a gradient of 5%CH₃CN (aq) to 75% CH₃CN (aq) buffered with 10 mM NH₄OAc. The eluent wasrun through a UV cell and then split between an ELSD detector and anAgilent MSD1100.

LCMS Method B: A 4×125 mm 5 uM Phenomenex Aqua column using a gradientof 5% CH₃CN (aq) to 75% CH₃CN (aq) buffered with 10 mM NH₄OAc. Theeluent was run through a UV cell and then split between an ELSD detectorand an Agilent MSD1100.

Example 1 Preparation of1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycin A

Amikacin sulfate 1.0 grams (Commercially available from a number ofchemical companies: O.M. Chemical Co., Ltd., Shanghai, China; ZhejiangWinsun Imp. & Exp. Co., Ltd, Zhejiang, China; Ningbo DHY PharmaceuticalCo., Zhejiang, China; and Shanghai Xudong Haipu Pharmaceutical Co., Ltd,Shanghai, China) was treated with 4 grams of Amberlite ion exchangeresin (OH— form, Aldrich, Milwaukee, Wis.) in 10 mL of water for 1 hour.The resultant solution was filtered and lyophilized to give AmikacinFree Base. The Amikacin free base (5.0 g, 8.5 mmoles) was dissolved in amixture of 1,4-dioxane (165 mL) and H₂O (235 mL). To this solution wasadded tert-butyloxycarbonyloxy-5-norbornene-2,3-dicarboxamide (1.3 g,7.8 mmoles) dissolved in a mixture of CH₂Cl₂ (10 mL) and 1,4-dioxane (5mL), over 50 min at room temperature. The reaction was stirred at roomtemp for 3 hours, and then evaporated under reduced pressure. Theresultant solid was subjected to silica gel chromatography (4:4:2, MeOH,CH₂Cl₂, NH₄OH, R_(f)=0.25) and then lyophilized to give the titlecompound, 1.6 g (30%) as a white solid.

¹H NMR (D₂O, 300 MHz) δ 5.45 (d, 1H, J=3.3 Hz), 5.05 (d, 1H, J=3.3 Hz),4.06 (m, 1H), 3.94 (m, 2H), 3.7 (m, 4H), 3.6-3.5 (m, 5H), 3.4-3.0 (m,7H), 1.99 (m, 1H), 1.87 (m, 1H), 1.66 (m, 1H), 1.52 (m, 1H), 1.34 (s,9H). ¹³C NMR (D₂O, 75 MHz) δ 175.3 (HABA, C═O), 157.2 (Boc, C═O), 97.0,96.3, 81.2, 80.1 (Boc, quaternary), 79.5, 73.1, 71.3, 71.0, 70.2, 70.0,68.3, 68.0, 67.7, 65.6, 58.9 (CH₂), 53.9, 48.2, 47.3, 39.4 (CH₂), 35.5(CH₂), 32.44 (CH₂), 31.7 (CH₂), 26.7. LCMS Method A; (retention time,m/z) 2.14 min, 686.2 (M+H) and 708.1 (M+Na). Starting material(R_(f)=0.10) was also recovered (1.7 g, 38%). In addition, one morecompound was recovered (R_(f)=0.74) and had a mass spectrum consistentwith6′-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA (1.47 g, 24%). LCMS Method A; (retention time, m/z) 2.68 min, 820.2(M+H) and 842.2 (M+Na).

Example 2 Preparation of6′-N-(benzyloxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA

1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycin A (2.4 g,3.5 mmoles) was dissolved in a mixture of 1,4-dioxane (9 mL) and water(9 mL) at room temperature. To this solution was addedbenzyloxycarbonyloxy-5-norbornene-2,3-dicarboxamide (1.0 g, 3.2 mmoles),dissolved in 1,4-dioxane (2 mL), over 20 minutes at room temperature.The reaction was stirred for 1 hour, and then evaporated under reducedpressure. The resultant solid was subjected to silica gel chromatography(6:2:1.5:0.5, CH₂Cl₂, MeOH, 1,4-dioxane, NH₄OH, R_(f)=0.36) and thenlyophilized to give the title compound 1.0 g (38%) as a white solid.

¹H NMR (DMSO-d₆/D₂O, 300 MHz) δ 7.4 (m, 5H), 5.04 (d, 1H, J=12.6 Hz),4.96 (d, 1H, J=12.6 Hz), 4.96 (d, 1H, J=3.3 Hz), 4.88 (d, 1H, J=3.3 Hz),3.83 (dd, 1H, J=9.3, 3.3 Hz), 3.8-3.6 (m, 5H), 3.6-3.35 (m, 7H),3.35-2.8 (m, 10H), 1.85 (m, 1H), 1.88 (m, 1H), 1.53 (m, 1H), 1.33 (s,9H), 1.17 (m, 1H). ¹³C NMR (DMSO-d₆/D₂O, 75 MHz) δ 173.8 (HABA, C═O),156.2, 155.6, (137.2, 128.3 and 127.7, Ph), 101.3, 97.5, 97.5, 80.0,77.5, 74.8, 72.8, 72.7, 72.2, 71.5, 71.4, 71.3, 69.2, 68.4, 60.2, 54.7,49.5, 48.7, 42.0 36.8, 35.5, 34.2, 28.2. LCMS Method A; (retention time,m/z) 2.68 min, 820.3 (M+H) and 842.2 (M+Na). One additional compound wasrecovered (R_(f)=0.51).

Example 3 Preparation of3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)-amino-2-S-hydroxybutyryl]kanamycinA

6′-N-(Benzyloxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA (0.99 g, 2.9 mmoles) was dissolved in 1,4-dioxane (15 mL)/H₂O (5 mL)and then treated dropwise with di-tert-butyl dicarbonate (0.78 g, 3.6mmoles) in 1,4-dioxane (3 mL) over 10 minutes at room temperature. Thereaction was stirred for 16 hours and then evaporated to give a whitesolid. The solid was suspended in tert-butylmethylether (30 mL) and thendecanted twice. The resultant solid (0.90 g, 73%) was used withoutfurther purification. TLC; (6:2:1.5:0.5, CH₂Cl₂, MeOH, 1,4-dioxane,NH₄OH, R_(f)=0.42). A portion of this solid (780 mg, 0.77 mmoles) wastreated with 10% Pd/C (70 mg) in 90% MeOH (60 mL) under a 10 psi H₂atmosphere for 3 hours to give a 93% yield of the title compound (627mg) as the HCl salt.

Example 4 General Procedure for Reductive Amination and Deprotection

3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycinA (120 mg, 0.135 mmoles) is dissolved in MeOH (2 mL),trimethylorthoformate (5 mL), tetrahydrofuran (5 mL) and acetic acid(400 mL). To this stirred mixture is added 1-4 equiv of an aldehyde(R₁—C(═O)H) with stirring for 1 hour followed by addition of Na(CN)BH₃(50 mg). The mixture is allowed to stir overnight, evaporated underreduced pressure and purified by flash chromatography (6:2:1:1.5:0.5,CH₂Cl₂, MeOH, 1,4-dioxane, NH₄OH). The product fractions are evaporatedunder reduced pressure and the residue is dried under reduced pressureovernight, and then treated with 1N HCl in dioxane (2 mL) for 1 hour.The solvent is decanted and the solid is washed with CH₃CN (4 mL×3) andsubsequently dried under reduced pressure to give the 6′-substitutedfully deprotected aminoglycoside as its HCl salt. In most of thesyntheses the R₁ group derives from the selected aldehyde R₁—C(═O)H andR₂ is H. In some cases it is possible to get disubstitution where R₁ andR₂═R₁ from the aldehyde but aside from the formaldehyde example givingR₁═R₂═CH₃ almost exclusively, the percentage of disubstitution isminimal and the monosubstituted compound can be purified by columnchromatography.

Example 5 Preparation of (IBIS00561961):6′-N,N-Dimethyl-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A, Formula I,R₁═R₂═CH₃

Using 112 mg of3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycinA and formaldehyde (4 equiv) and following the general procedure forreductive amination at the 6′-position illustrated above gives 35 mg(37%) of the title compound as the HCl salt. Formaldehyde is an exampleof an aldehyde that predominantly give disubstitution. Most otheraldehydes give mono substitution with a small percentage if any of thedisubstitution product.

LCMS Method A; (retention time, m/z) 0.4 min, 614.2 (M+H).

Example 6 Preparation of (IBIS00561962):6′-N-Phenpropylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,Formula I, R₁═Ph(CH₂)₃, R₂═H

Using 120 mg of3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycinA and phenpropanal (1 equiv) and following the general procedure forreductive amination at the 6′-position illustrated above gives 28 mg(24%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.4 min, 704.3 (M+H).

Example 7 Preparation of (IBIS00561960):6′-N-Phenethylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A, FormulaI, R₁=Ph(CH₂)₂, R₂═H

Using 152 mg of3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycinA and phenylacetaldehyde (1 equiv) and following the general procedurefor reductive amination at the 6′-position illustrated above gives 30 mg(27%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.4 min, 690.4 (M+H).

Example 8 Preparation of (IBIS00561963):6′-N-Isobutyl-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A, Formula I,R₁=(CH₃)₂CHCH₂, R₂═H

Using 99 mg of3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycinA and isobutyrylaldehyde (1 equiv) and following the general procedurefor reductive amination at the 6′-position illustrated above gives 40 mg(37%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.4 min, 642.6 (M+H).

Example 9 Preparation of6′-deoxy-6-azido-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,

Sodium azide (1.89, 2.9 mmol) was dissolved in 5 mL of water and to thisstirred solution, at room temperature, was added CH₂Cl₂ (6 mL) and thebiphasic solution was cooled to 0° C. Triflic anhydride (1.0 mL, 6.0mmol) in CH₂Cl₂ (6 mL) was added dropwise over 20 minutes and thesolution was stirred for an additional 2 hours at 25° C. The organiclayer was then separated and the aqueous phase extracted with CH₂Cl₂(2×50 mL). The combined organic layers were washed with NaHCO₃ until thegas evolution ceased, and the organic phase was separated and held forthe next step.3,3″-N,N-di-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]kanamycinA (2 mmoles) was treated with the above solution for 4 hours to give thetitle compound.

LCMS Method A; (retention time, m/z) 0.3 min, 612.2 (M+H).

Example 10 Preparation of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxy-carbonyl)amino-2-S-hydroxybutyryl]kanamycinA

Amikacin free base (5.0 g, 8.5 mmoles) was dissolved in a mixture of1,4-dioxane (25 mL), Et₃N (5 mL) and H₂O (25 mL). To this solution wasadded tert-butyloxycarbonylanhydride (43 mmoles) dissolved in a mixtureof CH₂Cl₂ (5 mL) and 1,4-dioxane (5 mL), over 50 minutes at roomtemperature. The reaction was stirred at room temperature overnight andevaporated under reduced pressure. The resultant solid was washedseveral times with Et₂O, and then dried to give a 99% yield oftetra-N—BOC-Amikacin.

LCMS Method A; (retention time, m/z) 2.14 min, 686.2 (M+H) and 708.1(M+Na).

Without further purification, the white solid was dissolved in drypyridine (50 mL), cooled in an ice water bath and then treated dropwisewith tosylchloride (1.8 g, 9.3 mmoles) dissolved in pyridine (5 mL). Thereaction was allowed to proceed for 5 hours and then quenched withNH₄Cl. Water (100 mL) was added and the product was extracted with EtOAc(3×100 mL). The organic layer was subsequently washed with brine (300mL), dried (Na₂SO₄), filtered and evaporated to dryness. The crude solidwas purified by flash chromatography (95:5, CH₂Cl₂:MeOH) to give 4.8 g(50%) of the title compound as a white solid.

LCMS Method A; (retention time, m/z) 3.35 min, 1041.2 (M+H-Boc).

Example 11 General Procedure for Tosylate Displacement and Deprotection

6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA (100 mg, 87.7 mmoles) is treated with 10-40 equiv of a selectednucleophile and EtOH (4 mL) in a sealed tube at 70° C. After 24 to 96hours, the reaction is cooled to room temperature, and then evaporatedto dryness. The resultant crude material is purified by flashchromatography (6:2:1:1.5:0.5, CH₂Cl₂, MeOH, 1,4-dioxane, NH₄OH). Theresultant solid is dried under reduced pressure overnight, and thentreated with 1N HCl in dioxane (2 mL) for 1 hour. At that time, thesolvent is decanted and the solid is washed with CH₃CN (4 mL×3) andsubsequently dried under reduced pressure to give the 6″-substitutedfully deprotected aminoglycoside as its HCl salt.

Example 12 Preparation of Example 1 (IBIS00561974):6″-deoxy-6″-methylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,R═N(H)CH₃

Using 100 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and methylamine (40 equiv) and following the general procedure fortosylate displacement and deprotection illustrated above gives 57 mg(63%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 599.2 (M+H).

Example 13 Preparation of Example 1 (IBIS00561977):6″-deoxy-6″-phenethylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,R═NH(CH₂)₂Ph

Using 100 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and phenethylamine (10 equiv) and following the general procedure fortosylate displacement and deprotection illustrated above gives 29 mg(29%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 690.2 (M+H).

Example 14 Preparation of Example 1 (IBIS00561980):6″-deoxy-6″-azido-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A, R═N₃

Using 152 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and sodium azide (10 equiv) and following the general procedure fortosylate displacement and deprotection illustrated above gives 120 mg(85%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 611.2 (M+H).

Example 15 Preparation of (IBIS00561976):6″-deoxy-6″-cyclohexylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,R=cyclohexylamine

Using 120 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and cyclohexylamine (20 equiv) and following the general procedure fortosylate displacement and deprotection illustrated above gives 89 mg(75%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 667.2 (M+H).

Example 16 Preparation of (IBIS00561975):6″-deoxy-6″-isopropylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,R═NHCH(CH₃)₂

Using 100 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and isopropylamine (20 equiv) and following the general procedure fortosylate displacement and deprotection illustrated above gives 77 mg(82%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 627.2 (M+H).

Example 17 Preparation of (IBIS00561982):6″-deoxy-6″-propylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycin A,R═NH(CH₂)₂CH₃

Using 109 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and propylamine (20 equiv) and following the general procedure fortosylate displacement and deprotection illustrated above gives 68 mg(72%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 627.2 (M+H).

Example 18 Preparation of (IBIS00561983):6″-deoxy-6″-dimethylaminoethylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycinA, R═NH(CH₂)₂N(CH₃)₂

Using 64 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and N,N-dimethylamino-ethylamine (10 equiv) and following the generalprocedure for tosylate displacement and deprotection illustrated abovegives 21 mg (21%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 656.3 (M+H).

Example 19 Preparation of (IBIS00561984):6″-deoxy-6″-benzylaminoethylamino-N-1-(4-amino-2-S-hydroxybutyryl)kanamycinA, R═NH(CH₂)₂NHBn

Using 99 mg of6′-Tosyl-6′,3,3″-N-(tert-butoxycarbonyl)-1-N-[4-(tert-butoxycarbonyl)amino-2-S-hydroxybutyryl]-kanamycinA and N-1-benzylamino-ethylamine (10 equiv) and following the generalprocedure for tosylate displacement and deprotection illustrated abovegives 37 mg (34%) of the title compound as the HCl salt.

LCMS Method A; (retention time, m/z) 0.3 min, 718.3 (M+H).

Example 20 Removal of 4-amino-2-S-hydroxybutyryl side chain(IBIS00561957): 6″-deoxy-6″-phenethylamino-kanamycin A

6″-deoxy-6″-phenethylamino-N-1-(4-amino-2-S-hydroxybutyryl) kanamycin A(100 mg) was refluxed in 2N NaOH for 1-2 days to give the titlecompound.

LCMS Method A; (retention time, m/z) 0.3 min, 589.2 (M+H).

Example 21 Assay to Determine the Minimum Inhibitory Concentrations(MICs, Bacteria)

The MIC assays are carried out in 150 μL volume in duplicate in 96-wellclear flat-bottom plates. The bacterial suspension from an overnightculture growth in appropriate medium is added to a solution of testcompound in 2.5% DMSO in water. Final bacterial inoculum isapproximately 10²-10³ CFU/well. The percentage growth of the bacteria intest wells relative to that observed for control wells containing nocompound is determined by measuring absorbance at 595 nm (A₅₉₅) after20-24 hours at 37° C. The MIC is determined as a range of concentrationwhere complete inhibition of growth is observed at the higherconcentration and bacterial cells are viable at the lower concentration.Both ampicillin and tetracycline are used as antibiotic positivecontrols in each screening assay. Both ampicillin and tetracycline areused as antibiotic-positive controls in each screening assay for S.pyogenes, E. coli imp-, E. coli, S. aureus, E. faecalis, K. pneumoniaeand P. vulgaris. Ciprofloxacin is used as an antibiotic positive controlin each screening assay for P. aeruginosa.

Selected compounds were assayed for antibacterial activity with theresult tabulated below:

P aeruginosa P aeruginosa Compound ID Comments P aeruginosa 25416 2924827853 IBIS00404962 Amikacin <0.625 1.25-2.5   0.6-1.25 IBIS00262400Kanamycin A 2.5-5   1.25-2.5  >40 IBIS00561960  5-10 2.5-5.0 >40IBIS00561961 >40 10-20 >40 IBIS00561962 2.5-5.0 2.5-5.0 >40IBIS00561963 >40 10-20 >40 IBIS00561957 NA NA NA IBIS00561975 10-2010-20  2.5-5.0 IBIS00561976  5-10 >40 >40 IBIS00561977  5-10 10-20  5-10IBIS00561974 0.625-1.25  2.5-5.0 1.25-2.5 IBIS00561980 0.625-1.25 2.5-5.0 1.25-2.5 IBIS00561982 2.5-5.0  5.0-10.0 1.25-2.5 IBIS005619831.25-2.5  1.25-2.5  1.25-2.5 IBIS00561984  5.0-10.0  5.0-10.0  5-10

P. vulgaris A. bauman11 E. coli S. aureus Compound ID 8427 WR-2 2592213709 IBIS00404962 <0.625 0.625-1.25  1.25-2.5  5-10 IBIS00262400 >40 0.6-1.25 >40 1.25-2.5  IBIS00561960 >40 2.5-5.0 >40 >40 IBIS0056196120-40 2.5-5.0 20-40  20-40  IBIS00561962 >40 1.25-2.5  >40 >40IBIS00561963 >40 2.5-5.0 >40 >40 IBIS00561957 NA NA >10 5.0-10.0IBIS00561975 10-20 1.25-2.5  10-20  10-20  IBIS00561976 >40 5-10 5-105-10 IBIS00561977 10-20 1.25-2.5  5-10 5-10 IBIS00561974 1.25-2.5 2.5-5.0 10-20  20-40  IBIS00561980 <0.625 2.5-5.0 2.5-5   5-10IBIS00561982 2.5-5.0  5.0-10.0 5-10 5-10 IBIS00561983 1.25-2.5  2.5-5.05-10 5-10 IBIS00561984 1.25-2.5  10.0-20.0 5-10 >40.

Example 22 Mass Spectrometry Based Binding Assay

Screening is performed by measuring the formation of non-covalentcomplexes between a single ligand or ligand mixture and the appropriateRNA target, along with suitable control structured RNA target(s)simultaneously using a 9.4 T FT-ICR mass spectrometer as detector. Fullexperimental details of the assay have been described in relatedliterature (Sannes-Lowery, et al. in TrAC, Trends Anal. Chem. 2000, 19,481-491 and Sannes-Lowery, et al. in Anal. Biochem. 2000, 280, 264-271.In a typical experiment, 10 μL of an aqueous solution containing 100 mMammonium acetate buffer, 2.5 or 5 μM of each RNA, and 33% isopropylalcohol (to aid ion desolvation) is prepared with differentconcentrations of each ligand or ligand mixture Samples are introducedinto the electrospray ionization source (negative ionization mode) at 1μL/min and ions are stored for 1 sec in an RF-only hexapole followingdesolvation. The abundances were integrated from the respective ions forfree RNA and the ligand-RNA complex. The primary (1:1 RNA:ligand) andsecondary (1:2 complex, if observed) KD values are determined bytitrating a single ligand through a concentration range of 0.25-25 μMwith an RNA target concentration of 0.10 μM. The peak ratios aremeasured at each concentration, then a plot of complex/free RNA versusconcentration of ligand added is fitted to a second (or higher) orderbinding polynomial to determine the KD.

Each reference cited herein, including but not limited to, patents,patent applications, patent publications, articles, treatises, andtexts, is hereby incorporated by reference in its entirety.

1. A compound of formula I:

wherein: each R₁ and R₂ is, independently, H or an amino protectinggroup; each R₃ is, independently, H or a hydroxyl protecting group; R₄and R₅ are each independently, H, amino protecting group, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl or substituted C₂-C₁₂ alkynyl; Q₁ is hydroxyl, aprotected hydroxyl, cyano, azido, or NR₈R₉; Q₂ is NH₂ or N(CH₂R₆)R₇; R₆and R₇ are each, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₇-C₉ alicyclic radical, substituted C₇-C₉ alicyclic radical,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl orsubstituted C₂-C₁₂ alkynyl; R₈ and R₉ are each, independently, H, CN,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical,substituted C₇-C₉ alicyclic radical, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl or substituted C₂-C₁₂ alkynyl; wherein saidsubstituted groups are mono or poly substituted with optionallyprotected substituent groups selected from halogen, trifluoromethyl,cyano, OJ₃, NJ₁J₂, C(═O)—NJ₁J₂, N(H)C(═O)-J₁, N(J₁)-(CH₂)_(n)—OJ₃,N(J₁)-(CH₂)_(n)—NJ₁J₂, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, C₇-C₉alicyclic radical, substituted C₇-C₉ alicyclic radical, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, azido, carboxy, acyl (C(═O)—X), ═O, cyano, sulfonyl(S(═O)₂—X) and sulfoxyl (S(═O)—X); each X is, independently, H, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl; each J₁ and J₂ is, independently, H,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—X), a heterocycle radical, asubstituted heterocycle radical, heteroaryl or substituted heteroaryl;each J₃ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or ahydroxyl protecting group; n is from 1 to 20; and with the proviso thatwhen Q₁ is hydroxyl or protected hydroxyl, Q₂ is N(CH₂R₆)R₇ and R₇ is Hthen R₆ is other than H or methyl.
 2. (canceled)
 3. The compound ofclaim 1 wherein each R₁ and R₂ is H.
 4. The compound of claim 1 whereineach R₃ is H.
 5. The compound of claim 1 wherein Q₁ is hydroxyl.
 6. Thecompound of claim 1 wherein Q₁ is NR₈R₉.
 7. The compound of claim 6wherein one of said R₈ and R₉ is H and the other of said R₈ and R₉ isC₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical orsubstituted C₇-C₉ alicyclic radical.
 8. (canceled)
 9. The compound ofclaim 1 wherein Q₁ is NH₂.
 10. The compound of claim 9 wherein R₇ is Hor C₁-C₁₂ alkyl.
 11. The compound of claim 9 wherein R₆ is C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₇-C₉ alicyclic radical or substituted C₇-C₉alicyclic radical.
 12. The compound of claim 11 wherein R₆ issubstituted C₁-C₁₂ alkyl wherein each of said substituents is,independently, C₇-C₉ alicyclic radical, substituted C₇-C₉ alicyclicradical, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical,substituted heterocycle radical, heteroaryl or substituted heteroaryl.13-14. (canceled)
 15. The compound of claim 1 wherein one of R₄ and R₅is H and the other of R₄ and R₅ is C₁-C₁₂ alkyl or substituted C₁-C₁₂alkyl. 16-17. (canceled)
 18. The compound of claim 1 wherein each of R₁,R₄ and R₅ are H.
 19. The compound of claim 18 wherein Q₁ is OH.
 20. Thecompound of claim 19 wherein R₇ is H.
 21. (canceled)
 22. The compound ofclaim 19 wherein R₆ is H and R₇ is CH₃.
 23. The compound of claim 18wherein Q₂ is NH₂.
 24. The compound of claim 23 wherein R₈ is H. 25.(canceled)
 26. The compound of claim 1 wherein Q₁ is azido or cyano.27-29. (canceled)
 30. The compound of claim 1 having the configuration:


31. The compound of claim 1 for use in therapy.